Soy proteins are valuable ingredients in foods due to their high nutritional value.
Soy proteins have found wide acceptance in Asia and are one of the principal sources of protein in a traditional Asian diet. Soy protein has not been as widely accepted in North America. Many American consumers object to the flavor of soy.
Raw soybeans and soy flour are characterized by odors described as green, grassy, bitter and beany and are therefore undesirable to many consumers. Volatile compounds contributing to soy flavor have been identified in numerous publications over the past 4 decades. A review of soy flavor (MacLeod, G. & Ames, J., (1988) Soy flavor and its improvement, CRC Critical Reviews in Food Science and Nutrition. 27 (4): 219-400) stated that 334 separate volatile compounds had been identified from soybeans, flours, concentrates, isolates, and textured soy proteins. The compounds identified were from the chemical classes of aliphatic hydrocarbons, alicyclic hydrocarbons, terpenoids, aliphatic alcohols, aliphatic aldehydes, aliphatic ketones, alicyclic ester, aliphatic ethers, aliphatic amines, aliphatic nitrile, chlorine containing compounds, benzenoids, sulfur compounds, benzenoids, sulfur compounds, furanoids, thiophenoids, pyrroles, pyridine, pyrazines, and thiazoles.
Specific compounds that have been identified as volatile components contributing to soy flavor include ethyl vinyl ketone, n-hexanol, n-pentanol, n-heptanol, methanol ethanol, ethanal, propanal, acetone, pentane, pentanal, hexanal, n-hexanal, acetaldehyde, acetone, and 2-heptenal.
Volatile components in soy products may be formed from precursors in the soybean. Factors affecting the formation of these compounds are oxygen tension, enzymes, temperature, moisture content and the possible presence of accelerators and/or inhibitors. Lipid oxidation and the effect of heating on carbohydrates and proteins have the greatest effect on the formation of volatile compounds. Minor factors contributing to flavor compounds include thermal decomposition of phenolic acids and thiamin and the degradation of carotenoids (MacLeod & Ames, supra).
Blade, R. J. (1990) Factors influencing endogenous flavor compounds in soybeans Ph.D. Dissertation, Clemson University identifies 21 volatile compounds in stored soybeans with gas chromatography-mass spectrometer analysis (GC-MS). Some compounds were not identified due to the inavailability of reference compounds or limitations in the sensitivity of the GC-MS. Predominant compounds isolated included: acetic acid, 1-hydroxy-2-propanone, butyrolactone, 1,3-dihydroxy-2-propanone, 2,6-dimethoxyphenol, 4-methylphenol, 3-hydroxy-4-methylacetophenone, palmitic acid, and stearic acid.
The undesirable flavor associated with soybeans and soy products has prompted research to develop methods to improve soy flavor. Past studies have focused on 3 main ideas: (1) Inhibition or inactivation of the lipoxygenase enzyme, (2) Removal of flavor compounds and precursors of flavor compounds, and (3) Masking the unwanted flavor (MacLeod & Ames, supra).
One area where soy protein has gained wide acceptance, despite its flavor, is in the production of infant formula. Formula such as Isomil.RTM., produced by the Ross Products Division of Abbott Laboratories, utilizes soy for the sole source of protein. Research has focused upon the removal of certain substances from the soy protein prior to its utilization in infant formula. These substances include nucleic acids, phytic acid (phytate), phytoestrogens, and the volatile substance described above. A commercially viable process for removing all of these substances has not been developed to date. Thus research efforts continue in the field
Phytic acid is inositol hexaphosphoric acid, and is part of a large class of compounds that influence the functional and nutritional properties of foods. The phytic acid content of soybeans is reported to be between 1.0 and 1.47% of the dry weight. This is about 60% of the total phosphorus in the soybean. The amount of phytic acid in soy flour has been reported to be as high as 2.24% (w/w). Phytate forms complexes with proteins and with mono- and divalent cations. Therefore, phytate in food components may cause the proteins and minerals to have limited bioavailability. Since phytate is associated with the proteins, protein products also have high levels of phytate.
Phytase is an enzyme that hydrolyzes phytic acid to myo-inisitol and inorganic phosphate. Phytases are special kinds of acid phosphatases that hydrolyze phosphate from phytic acid as well as other phosphorylated substrates. This enzyme is present in plants including seed and germinating beans. The use of phytase in soybeans is limited (Stutardi, Buckle, K. A. (1986) The characterisitic of soybean phytase, Journal of Food Biochemistry, (10: 197-216)).
Anno, T., Nakanishi, K., Matsuno, R., Kamikubo, T. (1985) Enzymatic elimination of phytate in soybean milk, Nippon Shokuhin Kogyo Gakkaishi, 32(3): 174-180, hydrolyzed phytate from soybean milk with free wheat phytase and immobilized phytase.
The phytic acid content in the soybean milk was from 0.52 to 1.11 mg/g. The optimum temperature of the phytase enzyme was from 45 to 50.degree. C. The optimum pH of the enzyme was 5.0 to 5.7. The enzyme was stable from pH 3.5 to 7.0. At pH lower than 6.0 the soybean proteins precipitated out. Phytate and protein interacted and formed stronger complexes at acidic pH. This interaction was found to decrease the solubility of the proteins, and influence the hydration, emulsifying properties and dispersibility.
Nucleic acids are another substance that would be desirable to remove from soy protein. Infant formula incorporating soy protein produced via current commercial processes has significantly higher levels of nucleic acids than human breast milk. Defatted soybeans reportedly contain 1.66% ribonucleic acid. Nucleotides contain a nitrogenous base (pyrimidine or purine), a pentose and a phosphate. A nucleoside is a nitrogenous base and a pentose without a phosphate (Lehnigher, A. L., Nelson, D. L., Cox, M. M. (1993) Principles of Biochemistry. New York: Worth Publishers)
Phytoestrogens occur in a variety of plants including soybeans. Phytoestrogens are defined as plant substances that are structurally and functionally similar to the gonadol steroid, 17 B-estradiol, that produce estrogenic effects. The desirability of phytoestrogens depends upon the age and sex of the individual who is consuming the soy protein. Phytoestrogens are highly desirable in menopausal and peri-menopausal females. The phytoestrogen mimics the estrogen which the female is either no longer producing or is producing in much smaller amounts.
By contrast, since phytoestrogens are not found in human breast milk it would be desirable to minimize their levels in infant formula. A detailed review of the effects of phytoestrogens on mammals is reported by Kaldas and Hughes in Reproductive and General Metabolic Effects of Phytoestrogens in Mammals, Reproductive Toxicology, Vol. 3, pages 81-89 (1989).
As used herein, the terms "phytoestrogens" and "isoflavones" should be considered interchangable. The term "isoflavones" refers to the compounds having the following general formula, with specific compounds identified in Table 1. ##STR1##
TABLE 1 Chemical structures of isoflavones found in soybeans Isoflavone R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 Daidzein H H OH OH H Genistein OH H OH OH H Glycitein H OCH.sub.3 OH OH H Daidzin H H O-glucoside OH H Genistin OH H O-glucoside OH H Glycitin H OCH.sub.3 O-glucoside OH H
As described in Table 2, daidzin, genistin and glycitin are the beta-glucoside conjugate (glucones) moieties. Daidzein, genistein and glycitein are the unconjugated (aglucones) moieties. As used herein "daidzein, genistein and glycitein levels" include both the conjugated and unconjugated moieties. The typical ratio of genistein to daidzein to glycitein in soy protein is 67 to 30 to 3.
Soy proteins are typically in one of three forms when consumed by humans. These include flour (grits), concentrates, and isolates. All three types are made from defatted soybean flakes. Flours and grits contain at least 50% protein and are prepared by milling the flakes. Soy protein concentrates contain at least 70% protein on a dry weight basis. Concentrates are made by repeatedly washing the soy flakes with water, which may optionally contain low levels of food grade alcohols or buffers. The effluent from the repeated washings is discarded and the solid residue is dried, thereby producing the desired concentrate. The yield of concentrates from the starting material is approximately 60-70%.
Soy protein isolates contain a minimum of 90% protein on a dry weight basis. Isolates are made by extracting the soy flour with a dilute alkali (pH &lt;9) and centrifuging. The extract is adjusted to pH 4.5 with a food grade acid such as sulfuric, hydrochloric, phosphoric or acetic acid. At a pH of 4.5, the solubility of the proteins are at a minimum so they will precipitate out. The acid precipitated protein curd is centrifuged, washed, neutralized and spray dried to produce the soy protein isolate. The yield of the isolate is 30% of the original soy flour and 60% of the protein in the flour.
Due to the potential for improving the properties of soy protein, research has been carried out on alternative ways of preparing soy flours, concentrates and isolates. Some of this research has focused upon ultrafiltration. Ultrafiltration is a method used to separate molecules based on molecular size or shape. The membrane acts as a selective barrier. A solution is pumped through a semi-permeable membrane. The membrane retains compounds higher in molecular weight while smaller molecules and water pass through the membrane.
Due to the pressure gradient across the membrane, smaller molecules and water are forced through the membrane. This is referred to as the permeate. Larger molecules (macromolecules) remain in the membrane and are circulated through the system. This is referred to as the retentate. Ultrafiltration retains particles in the range of 0.10 mm to 10 mm (Cheryan, M. (1986) Ultrafiltration Handbook. Lancaster, Pa.: Technomic Publishing Co., Inc.).
The first studies applying membrane filtration systems to soy protein separation began in the early to mid 1970s. Membrane filtration processing of soy products seemed promising due to the ability to separate the large protein fractions from the smaller unwanted phytate and oligosaccharide molecules (Omasaiye, O., Cheryan, M., Matthews (1979b) Ultrafiltration of soybean water extracts: Processing characteristics and yields. Journal of Food Science. 44: 1027-1031).
Okubo, K., Waldrop, A. B., Lacobucci, G. A., Myers, D. V. (1975) Preparation of low-phytate soybean protein isolate and concentrate by ultrafiltration, Cereal Chemistry, 52: 263-271, produced a low-phytate soybean protein isolate using an ultrafiltration method. The first step was to remove the phytate from the soybean with an extraction procedure and dialysis. The next step was continuous diafiltration. Three different methods were used to prepare the isolates for diafiltration. The first was maintained at pH 8.5 at 65.degree. C. with EDTA added. The temperature and pH of the second sample were maintained in the range of the optimum temperature for plant phytases (pH 5.5 and 55.degree. C.). The third sample was maintained at pH 3 and 25.degree. C. with added calcium. Phytate removal occurred at pH 5.5 and 55.degree. C. and at pH 3 with calcium. The authors report the most effective removal of phytate in the soy protein isolate occurred at a pH of 3.0 with added calcium.
Okubu's process did not use ultrafiltration to remove the phytate and suffered from the disadvantage of subjecting the soy to an acidification step. Acidification results in denaturation of the protein which decreases the functionality of the soy. The capability of the denatured soy to serve as an emulsifier is decreased. Proteins typically serve as an emulsifier in enteral formula. They assist in stabilizing the emulsion.
Omasaiye, O., Cheryan, M., Matthew, E., (1978) Removal of oligosaccharides from soybean water extracts by ultrafiltration, Journal of Food Science, 43: 354-360, made a full-fat soy protein concentrate by ultrafiltration. Defatted soy flour was the typical starting material for soy protein concentration processing. In this study, soybeans were the starting material. Soybean water extracts were fed into the ultrafiltration system for continuous diafiltration. The composition of the diafiltered product was 58.26% protein, 33.56% fat, 0.77% oligosaccharides, 3.43% ash and 3.98% other compounds.
Omosaiye and Cheryan (1979b), supra, reported on the characteristics of soybean components such as protein, fat and ash during ultrafiltration. Water extracts of whole soybeans were produced by a process using the following steps: Soaking, blanching, grinding, filtration, and rinsing. The filtrate was used as the feed for the ultrafiltration system. A 50,000 molecular weight cut-off membrane was used and essentially no protein or fat was found in the permeate. Ash increased in the retentate as concentration increased, indicating some mineral binding to proteins since minerals should have been freely permeable to the membrane. The final product is this study contained 59.7% protein, 34.2% fat, 2.85% ash, 0.64% oligosaccharides and 0.065% phytic acid.
In an additional study, Omosaiye, O., Cheryan, M., (1979a) Low-phytate, full-fat soy protein product by ultrafiltration of aqueous extracts of whole soybeans, Cereal Chemistry, 56(2): 58-62, used a two step process which included ultrafiltration to produce a soy protein isolate low in phytic acid. The first step consisted of extracting the beans. This extract was the subjected to ultrafiltration. The phytate removal depended on the pH of the ultrafiltration solution. The greatest phytate removal occurred at pH 6.7. Less phytate was removed at pH 2.0, pH 8.0 and pH 10.0. These results may in part be explained by phytate-protein interactions. At pH 6.7, the phytate appeared to be water soluble, did not have a strong electrostatic attraction and the salt linkages were weak. The optimum pH for phytate removal was found to be the same as the pH for protein water extracts.
Nicholas, D. J., Cheryan, M. (1981) Production of soy isolates by ultrafiltration: Factors affecting yield and composition, Journal of Food Science, 46: 367-372, studied the factors affecting the yield and composition of soy protein isolates during an ultrafiltration process. The starting material was an extract of defatted soy flour. The molecular weight cut-off of the membrance was 50,000. In order for the ultrafiltration step to produce a product with a protein content of 90%, over 80% of the non-protein solutes needed to be removed. The starting material had a protein content of 65%. The highest protein content obtained was 84% on a dry weight basis. Therefore, the ultrafiltration step did not fractionate the compounds to the degree necessary to produce a soy protein isolate. Pumping problems and severe membrance fouling were sited as problems. As observed in other studies (Omosaiye and Cheryan, (1979b), supra) the mineral content did not decrease according to predicted permeability of the membrane, perhaps due to mineral-protein binding. The highest protein yield obtained was 86%.
In summary, the prior art shows that attempts have been made to: 1) produce soy protein isolates and concentrates utilizing ultrafiltration, and; 2) to remove phytate and volatiles from soy proteins. Such attempts have met with limited success. Several authors report severe fouling of the filtration membranes. Fouling is the build up of substances on the surface of the membrane. This prevents the membrane from performing its function of separating molecules on the basis of size. The presence of complex polysaccharides of large molecular weight has often been cited as the source of the fouling. The complex interaction between phytate and protein has been a further source of difficulty. Authors have reported using acid treatments prior to ultrafiltration to disrupt this interaction. The acidification however leads to a partial denaturation of the protein with corresponding adverse effects on its performance.
Thus a need exists in the art for a ultrafiltration process that can be used to produce soy protein on a commercial scale. A further need exist in the art for soy protein having reduced levels of phytoestrogens, phytate, and nucleic acids. A further need exists for a process for producing soy proteins isolates and concentrates that does not subject to the soy proteins to acidic conditions, since such conditions produce a partial denaturization of the protein.